Quinoline carboxamide core moiety-based compounds inhibit P. falciparum falcipain-2: Design, synthesis and antimalarial efficacy studies

Highlights

• A set of 25 quinoline carboxamide-based compounds was designed and synthesized to inhibit P. falciparum FP2.

• Integration of molecular hybridization strategy with in silico drug design was adopted.

• Compounds Qs17, Qs18, Qs20 and Qs21 displayed best results in docking and in vitro FP2 inhibition.

• These compounds inhibited P. falciparum growth with IC₅₀ values: 1.05, 1.95, 1.43 and 0.81 µM, respectively.

• Morphological and food-vacuole abnormalities much better than E-64 were observed.

Abstract

Targeting Falcipain-2 (FP2) for the development of antimalarials is a promising and established concept in antimalarial drug discovery and development. FP2, a member of papain-family cysteine protease of the malaria parasite Plasmodium falciparum holds an important role in hemoglobin degradation pathway. A new series of quinoline carboxamide-based compounds was designed, synthesized and evaluated for antimalarial activity. We integrated molecular hybridization strategy with in-silico drug design to develop FP2 inhibitors. In-vitro results of FP2 inhibition by Qs17, Qs18, Qs20 and Qs21 were found to be in low micromolar range with IC₅₀ 4.78, 7.37, 2.14 and 2.64 µM, respectively. Among the 25 synthesized compounds, four compounds showed significant antimalarial activities. These compounds also depicted morphological and food-vacuole abnormalities much better than that of E-64, an established FP2 inhibitor. Overall these aromatic substituted quinoline carboxamides can serve as promising leads for the development of novel antimalarial agents.

Introduction

Malaria is one of the most life menacing neglected disease caused by protozoan parasites of genus plasmodium. Approximately two hundred million infections and 438,000 deaths in a year, predominantly children have been reported by WHO due to malaria [1]. Majority of the antimalarial drugs reported till date target the asexual blood-stages of the parasite where it replicates within human erythrocytes [2]. However, liver- and transmission-stage of the parasite do not cause malaria symptoms. Due to the complex life cycle of the parasite and increasing emergence of resistance against conventional antimalarials and artemisinin combination therapy, malaria remains a challenging issue for the humanity. The identification of new apt drug targets and the development of effective anti-malarial molecules that target important parasite functions with a potential to act against multi-drug-resistant strains of the parasite is an indispensable need to prevent malaria pathogenesis and its transmission [3].

Proteases are among the attractive drug targets for the development of antimalarial therapy. Falcipain-2 (FP2) and falcipain-3 (FP3) are papain-family cysteine proteases of erythrocytic stages of P. falciparum that localize to the food vacuole and readily hydrolyze hemoglobin [4], [5]. Disruption of the FP2 gene led to the accumulation of undegraded hemoglobin in trophozoites, confirming a critical role of this enzyme in hemoglobin hydrolysis [6]. Inhibition of FP2 and related proteases led to the inhibition of parasite development [7], [8] and cured mice with murine malaria [9], [10].

E64 (Fig. 1), an irreversible inhibitor of cysteine proteases, displayed a significant outcome on growth, adherence and viability of parasite trophozoites [11]. Chiyanzu et al. [12] designed a new class of thiosemicarbazones with isatin scaffold as FP2 inhibitors. The most promising FP2 inhibitor of this series (Fig. 1a) showed an inhibition with IC₅₀ 4.4 µM. From such results, chalcones proved to be the robust inhibitors of cysteine proteases. Firstly, Li and co-workers in 1995 started working over the development of antimalarial chalcones as one of the derivatives: 1-(2,5-dichlorophenyl)-3(4-quinolinyl)- 2-propen-1-one (Fig. 1b) showed an IC₅₀ value of 200 nM against both CQ-resistant strain (W2) and CQ-sensitive strain (D6) of P. falciparum [13], [14]. Several peptide-based antitypanosomal agents such as compound c (Fig. 1c) have been found to inhibit FP2 of cysteine ​​protease rhodesain and P. falciparum of brucei rhodesiense.

Recently, (DDD107498), a quinoline-4-carboxamide has been reported with excellent pharmacokinetic and antimalarial properties, including activity against multiple life-cycle stages of the malaria parasite [15]. Compound SC81458 and the clinical development candidate, SC83288 (Fig. 2), both containing sulfonamide and piperazine groups, showed noteworthy results to cure a P. falciparum infection with tolerable toxicity. Properties like quick parasite killing, good safety margin, a potentially different mode of action and a distinct chemotype support SC83288 for the clinical trials against malaria [16]. The quinoline-4-carboxlic acid and it's analogs show diverse range of therapeutic activity such as antimalarial, antifungal and anti-leishmanial effects [17], [18].

Dual inhibitors of FP2 and FP3 have been identified through virtual screening of 241,000 compounds against homology models of FP2 and FP3 in three consecutive stages of docking [19]. Benzothiazole containing sulfonyl-2-nitrobenzene based compounds, for example compound 1 (Fig. 2) have been found to inhibit FP2 with IC₅₀ 11.14 μM. These compounds have been prophesied to bury into the S2 pockets of FP2 and FP3, thereby inhibit both the enzymes significantly [20]. Same way, potential antimalarial agents containing 4-aminoquinoline with natural product isatin scaffold as in compound 2 (Fig. 2) have been discovered with IC₅₀ 1.3–0.079 and 2.0–0.050 µM against a chloroquine-sensitive (D10) and two resistants (K1 and W2) strains of P. falciparum. Two such isatin based compounds 3 and 4 (Fig. 2) displayed invitro activity against K1 and W2 strains with IC₅₀ values of 51 and 54 nM, respectively. In addition, these compounds were found to inhibit parasitic cysteine protease FP2 [21]. Keeping these things into consideration, we attempted to incorporate different functional units of reported FP2 inhibitors and different clinical candidate antimalarials in one molecule as represented in (Fig. 2). Integration of structure based drug design technique and molecular hybridization was used to incorporate potential scaffolds in the designed molecules.

Here, we report the design, synthesis and biological evaluation of quinoline-4-carboxamide based analogues as FP2 inhibitors. The core moiety quinoline was prepared from isatin and acetophenone (Scheme 1) using the Pfitzinger reaction. We utilized diversity-oriented route to design and synthesize the target molecules. The 25 synthesized molecules were identified as potential inhibitors of FP2, among which the best compound QS20 displayed FP2 inhibition with IC₅₀ = 2.14 μM. In addition, the anti-plasmodial activity of these molecules against P. falciparum was tested in which QS20 exhibited better parasite inhibition with IC₅₀ = 0.81 μM.